In vitro development of Brugia pahangi and Brugia malayi in cultured mosquito thoraces.

In vitro cultivation of Brugia pahangi and subperiodic Brugia malayi one-day old larvae to infective stage larvae (L3) within thoraces excised from Aedes aegypti (Black Eye, Liverpool) and Anopheles quadrimaculatus was attempted. The mosquito thoraces were excised under aseptic conditions, 24 h after a blood meal on either B. pahangi- or B. malayi-infected jirds. The excised thoraces were washed aseptically and inoculated into a diphasic media. A nutrient agar base was overlaid with either Grace's insect cell culture medium or Schneider's Drosophila medium or a mixture (1:1) of these two media. Each overlay medium contained a 1 x concentration of antibiotic/antimycotic mixture plus 20% fetal bovine serum. The excised thoraces provided the intracellular milieu for development of Brugia larvae. In Grace's or Schneider's insect tissue culture medium alone, the filaria larvae of both species developed only to the second larval stage after 12 days; whereas, in a mixture (1:1) of Grace's and Schneider's media, some one-day old larvae of both Brugia species developed to the infective larval (L3) stage after 12 days. However, large numbers of both species of larvae developed to the infective larval stage when, prior to providing an infective blood meal, the mosquitoes of both species were fed 1 x concentration of antibiotic/antimycotic mixture in a 10% sucrose solution containing 0.1% p-aminobenzoic acid for 6 days. These results showed for the first time that if one-day old Brugia larvae are confined intracellularly in excised thoraces, they can then develop in insect tissue culture media without adding a feeder layer of mosquito cells or conditioning the media with mosquito cell lines.     

The effect of p-aminobenzoic acid and folic acid on the development of infective larvae of Brugia malayi in Aedes aegypti

Adult Aedes aegypti mosquitoes, infected with the subperiodic Brugia malayi, were found to enhance the development of the filarial parasites to the infective stage when they were exposed to a cotton pad soaked in 10% sucrose solution containing p-aminobenzoic acid (PABA) in 0.001, 0.005, 0.01, 0.05 and 0.1% concentrations. Similarly, larval development increased when the mosquitoes were fed with folic acid at 0.001, 0.01 and 0.1% concentrations. This stimulation was more when PABA or folic acid was given prior to the infected blood meal through the developmental period of the larvae. The data thus suggest that PABA and folic acid are nutrients for the development of B. malayi-microfilariae to the infective stage in A. aegypti.     

Experimental infection of subperiodic Brugia malayi in laboratory rats with emphasis on evaluation by four techniques

Infective larvae of subperiodic B. malayi from South Kalimantan (Borneo), Indonesia collected from laboratory-raised Ae. togoi mosquitoes after feeding on infected mongolian gerbils (Meriones unguiculatus) were inoculated subcutaneously into the groin areas of 15 SD and 36 LE rats. Blood was examined weekly by membrane filtration and thick smears starting 10 weeks post-infection. Microfilariae were found in 3 SD and 4 LE rats, the mf infection rate of 20% and 11% respectively. The prepatent period was significantly shorter in the SD rats (99-112 days) than those in the LE rats (110-153 days). The patent period was longer in the LE rats (208-703 days) than in the SD rats (236-543 days), and the mf density was similar (17.5 mf/20 c.mm blood against 16 mf/20 c.mm blood). At necropsy, 6 (3 female and 3 male) adult worms were recovered from 3 of 6 SD rats and 12 (9 female and 3 male) adult worms from 4 of 20 LE rats; all worms were found in the testes. The results of xenodiagnostic, histochemical staining and measuring spicules and protuberances, demonstrated clearly the difference between both species of Brugia. All dissected Ar. subalbatus mosquitoes exposed to B. pahangi became infected (100%), but none of those to subperiodic B. malayi were infected (0%). The mf of both species of Brugia in thick films stained with naphthol-AS-TR-phosphate showed that the excretory and anal pores of subperiodic B. malayi mf exhibited acid phosphatase activity and only a little activity was seen in other parts; while B. pahangi mf showed heavy diffuse acid phosphatase activity along the entire length of the body.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laboratory transmission of lymphatic filariasis by vector mosquitoes

Aedes togoi and Ae. aegypti were used to examine the transmission potential of Brugia pahangi to one of its natural hosts, the domestic cat. Although a larger proportion of microfilariae taken in by Ae. togoi developed into infective larvae, the total number of B. pahangi larvae recovered from a cat exposed to Ae. aegypti was larger than from a cat exposed to Ae. togoi. Factors influencing the transmission dynamics included: development of microfilariae to infective larvae; survival of mosquitoes; willingness to take repeated blood meals; and proportion of infective larvae that egress from mosquitoes during the feeding process. From 19 to 25% of infective larvae were transferred to a susceptible host. The feasibility of using a Brugia-cat model to do comparative vector efficiency studies was demonstrated.     

Infective larvae of Brugia: escape from mosquitoes into water and subsequent oral infectivity in jirds.

Published work showed that third-stage larvae (L-3s) escape into water from dead or dying, Brugia pahangi-infected, Aedes aegypti. The present study revealed the same escape phenomenon among B. pahangi-infected Armigeres subalbatus, Anopheles quadrimaculatus, and Aedes togoi, and among Brugia malayi-infected Ae. aegypti and Ae. togoi. L-3s maintained in water or in Lum's solution for 3 hours retained infectivity when tested in orally or subcutaneously exposed jirds; furthermore, L-3s recovered from mosquitoes dead for 24 to 48 hours were also infective by either portal of entry in jirds. Since L-3s may escape and remain infective in the field, it is conceivable that natural filarial infections might thus be acquired orally by definitive hosts.     

A comparison of host responses of the Mongolian jird to infections of Brugia malayi and B. pahangi

Host responses of jirds receiving a single subcutaneous inoculation of subperiodic Brugia malayi were compared with those of jirds similarly infected with B. pahangi. Parasite burdens, lymphatic lesion severity, granulomatous reactivity, antibody responses to parasite antigens, and complete blood cell counts were assessed at 60 and 150 days post-inoculation. At 60 days post-inoculation, percentages of adults recovered at necropsy and lymphatic lesion severity were greater in B. pahangi-infected jirds. At 150 days post-inoculation, lesion severity and percentages of worms recovered were similar in both infections. No significant differences were noted in either infection in reactivity to homologous or heterologous parasite antigens in any parameter measured. Similarities in the kinetics of the inflammatory reactivities of the 2 infections suggest that previous observations made in the jird-B. pahangi model could be utilized in designing studies using B. malayi. Further, the more marked lesion severity observed in B. pahangi-infected jirds and the relative ease of maintaining B. pahangi in the laboratory support the continued use of this system as a conceptual model for the study of lymphatic lesion pathogenesis.     

The influence of the gene sb in Culex pipiens on the development of sub-periodic Brugia malayi and Wuchereria bancrofti

The gene sb (filarial susceptibility, Brugia pahangi) in Culex pipiens controls the development also of sub-periodic B. malayi, but has no influence on the development of periodic Wuchereria bancrofti (Ceylon strain). C.p. fatigans (Kuala Lumpur), C.p. molestus (London) and Aedes aegypti (re fm strain) were all susceptible to the Ceylon strain of W. bancrofti, with susceptibility rate of 90.3%, 92.9% and 52.6% respectively. However, a low proportion of the larvae in A. aegypti developed to maturity, and this mosquito is less well adapted to W. bancrofti than is C. pipiens.     

The effect of 5-fluorouracil and 5-fluorocytosine on the development of the filarial nematodes Brugia pahangi and Dirofilaria immitis

5-fluorouracil and 5-fluorodeoxyuridine at 30 mg/kg body weight daily for four days inhibit microfilarial production in Brugia pahangi in the jird. Disruption of intrauterine embryogenesis was observed in treated female worms but the compounds were not macrofilaricidal or microfilaricidal under the conditions employed. 5-fluorocytosine possessed no filaricidal or embryostatic activity. The inhibition of microfilaria production by 5-fluorouracil was temporary and larval production was resumed within nine weeks. The compound also inhibited the development of B. pahangi and Dirofilaria immitis larvae in the mosquito Aedes aegypti, when administered to cages of mosquitoes as a 0.01 or 0.001% solution in a 10% aqueous sucrose solution on cotton wool wicks. The development of infective larvae of B. pahangi in the jird was inhibited by 5-fluorouracil.     

Radiolabeling of Brugia malayi infective larvae in mosquitoes with 75Se-methionine and detection of these larvae in tissues of the Mongolian jird by autoradiography

Through preliminary experiments, an effective method for radiolabeling Brugia malayi-infected mosquitoes in order to produce labeled infective Brugia larvae was developed. Starting on the 6th day after the infective blood meal, mosquitoes were fed a 7% sucrose solution containing 100 microCi/ml 75Se-L-methionine for 5 days. Infective larvae, retrieved 2 days after this labeling period, averaged 381 +/- 136 counts/min. Jirds were infected with these infective, labeled larvae either by allowing infected mosquitoes to feed on uninfected jirds for 30 min or by inoculating jirds subcutaneously in the groin with washed larvae recovered from mosquitoes. Jirds were killed at various times after infection and were sliced into approximately 0.5 mm thick sagittal sections, which were dried and placed on X-ray film. Autoradiograms were developed after 30-60 days at 5 degrees C. In a sample of 26 inoculated jirds, approximately 30% of the infecting larvae could subsequently be accounted for as Ag degrees foci on autoradiograms. The Ag degrees foci representing larvae were apparent up to 2.5 weeks after infection. In jirds infected by mosquito feeding, the Ag degrees associated with the feeding site persisted for more than 6 weeks after infection.     

The exsheathment of Brugia pahangi microfilariae under controlled conditions in vitro

Two reproducible techniques for the exsheathment in vitro of microfilariae of Brugia pahangi, and other sheathed microfilariae, are described. Microfilariae were isolated from infected cat blood by filtration and suspended in Hank's Balanced Salt Solution. The first technique involved the incubation of isolated microfilariae for one hour in 20 mM CaCl2 in a phosphate-free Balanced Salt Solution, during which time approximately 90% of the microfilariae lost their sheaths. The second method of exsheathing microfilariae of B. pahangi involved exposure of microfilariae to solutions of endopeptidase (5.8 units/ml) or papaya extract protease (3.0 units/ml) in Ca2+-free HBSS. Exsheathment rates of 95--100% occurred within 30 minutes in both enzyme solutions. Both the Ca2+ ion and the endopeptidase technique have proven equally effective in stimulating exsheathment of microfilariae of Brugia malayi, Wuchereria bancrofti and Litomosoides carinii. Such artificially exsheathed microfilariae are used for in vitro cultivation studies. The viability of Ca2+- and endopeptidase-exsheathed microfilariae of B. pahangi has been confirmed by inoculation of exsheathed larvae into susceptible female mosquitoes.     

Exsheathment of microfilariae of Brugia pahangi in the susceptible and refractory strains of Aedes aegypti

Exsheathment of microfilariae of Brugia pahangi was studied in susceptible (Liverpool) and refractory (Bora-Bora) strains of Aedes aegypti. It was found that the microfilariae tend to carry their sheaths into the haemocoel of both strains of Ae. aegypti within two hours after the engorgement of mosquitoes from a rat parasitized by filariae. The percentage of sheathed microfilariae in the haemocoel then progressively decreased to 0% at eight hours and to 1% at 24 hours post-ingestion in the Bora-Bora and Liverpool strains, respectively. Those microfilariae that remained in the midgut more than two hours after ingestion were most likely to cast off their sheaths there. The percentage of microfilariae exsheathed in the midgut progressively increased to about 91 and 78% at 24 hours post-ingestion in the Bora-Bora and Liverpool strains, respectively. These results suggested that the exsheathment of microfilariae occurs both in the haemocoel and in the midgut of two strains of Ae. aegypti.     

Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi.

A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross-hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved by Alu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% homologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes.     

Phosphorylcholine-bearing antigens in filarial nematode parasites: analysis of somatic extracts, in-vitro secretions and infection sera from Brugia malayi and B. pahangi

A set of cross-reactive antigens is described which are present in somatic extracts and in-vitro secretions of the filarial nematodes Brugia pahangi and B. malayi. A monoclonal antibody reactive with a repeating epitope on these molecules readily detects circulating antigen in the serum of animals infected with lymphatic filariae, using either an immunoradiometric assay or enzyme-linked immunosorbent assay (ELISA). This epitope has the immunological reactivity and chemical characteristics of the phosphorylcholine (PC) hapten. The anti-PC monoclonal has been used to define the antigens bearing this epitope, and in chromatographic studies on material from extracts of Brugia adult worms, a heterogeneous profile of PC-positive molecules are found. In sera from Brugia-infected jirds, an antigen with a native molecular weight of approximately 500,000 is observed, which displays limited sensitivity to protease degradation. However, denatured samples on Western blots show a major parasite circulating antigen of Mr 90,000. The detection of this antigen in the presence of excess host antibody is also demonstrated, taking advantage of the stability of the target epitope to a range of treatments designed to dissociate and eliminate immune complexes.     

Brugia malayi: ivermectin inhibits the exsheathment of microfilariae.

Brugia malayi-infected microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg of body weight injected subcutaneously. Susceptible Aedes aegypti mosquitoes were fed on treated jirds 24 hours later. Mosquitoes fed on untreated jirds served as controls. Infected mosquitoes were dissected at 1, 3, 24, 48, 72, and 96 hr after the blood meal, and differential counts of sheathed microfilariae, exsheathed microfilariae, and cast sheaths were performed using fluoresceinated wheat germ agglutinin. Microfilariae failed to exsheath in mosquitoes fed on ivermectin-treated jirds. Microfilariae from ivermectin-treated jirds also did not exsheath in vitro in the presence of 10 mM CaCl2, whereas 85-90% of sheathed microfilariae from untreated jirds exsheathed in vitro. In addition, sheathed microfilariae from untreated jirds, when pretreated in vitro with ivermectin at 0.25, 0.5, or 1 microgram/ml, lost their ability to exsheath in vitro in the presence of 10 mM CaCl2. However, ivermectin treatment had no effect on exsheathing of microfilariae when incubated with papaya protease. Thus, ivermectin appears to inhibit the intrinsic exsheathing process of microfilariae in the mosquito host, thereby blocking their development and further transmission of infection.     

应用DNA探针检测蚊体内丝虫幼虫的研究

本文用人工合成的寡核苷酸探针，经斑点杂交法检测蚊体内马来丝虫幼虫。可测到丝虫和微丝蚴２ｎｇ的ＤＮＡ量，不与其它动物丝虫标本发生交叉反应。将单个蚊直接压于硝酸纤维素膜上进行检测，感染蚊中含有１条感染期幼虫就可出现阳性反应。将一组蚊虫在裂解液中研磨集体检测时，在２０只蚊虫中有１只感染蚊即可检出。显示该探针可用于马来丝虫地区的蚊媒监测。     

Eosinophil hyporesponse of jirds induced by microfilariae of Brugia pahangi

Male jirds (Meriones unguiculatus) were inoculated sc with 100 infective larvae of Brugia pahangi. After 16 weeks, the animals were reinoculated with a comparable number of organisms. Blood eosinophil responses during the 5 weeks subsequent to this attempt to reinfect were much lower than those of comparable naive animals, while the response to a heterologous infection (Toxocara canis) was comparable to that of controls. Mebendazole was given to infected animals for 2 weeks beginning 5 weeks (prepatent) or 16 weeks (patent) after infection. At comparable intervals after drug administration, the animals were reinoculated with infective larvae and the blood eosinophil response was measured over a 5 week period. The response in the animals treated during the prepatent period was higher than the untreated infected controls. Treatment during the patent period had no demonstrable effect. Jirds made artificially microfilaremic by intravenous inoculation of viable filaria before and after the standard infecting dose had a low eosinophil response to infective larvae. A primary experience of jirds with the microfilariae of B. pahangi evokes an eosinophil response. Subsequent inoculation of larvae did not produce a comparable response.     

Cryopreservation of third-stage larvae of Brugia malayi and Dipetalonema viteae

Methods are described for the cryopreservation of third-stage larvae of Brugia malayi. Optimum conditions utilized larvae free from the mosquito host frozen at the rate of -1 degree or -0.8 degrees C per min in medium containing 9% dimethyl sulfoxide and 0.004 M polyvinylpyrrolidone. Nonfrozen or thawed larvae were inoculated intraperitoneally into jirds (Meriones unguiculatus), the thawed larvae after cryogenic storage for 5-378 days. In general, the percentage of adult worms recovered at necropsy was comparable between the two groups and ranged from a mean of 6-9% of the larval inoculum. In addition, three of four patas monkeys (Erythrocebus patas) inoculated with thawed B. malayi larvae developed patent infections. The cryopreservation of third-stage larvae of Dipetalonema viteae also is discussed.     

Identification of Brugia malayi in vectors with a species-specific DNA probe

We evaluated the potential value of a cloned sequence of genomic DNA of Brugia malayi as a species-specific probe. Clone pBm 15 reacted with all stages of 8 different geographic isolates of B. malayi and cross-hybridized with microfilariae of B. timori. It did not hybridize with Wuchereria bancrofti or with B. pahangi, W. kalimantani, Dirofilaria repens, Breinlia booliati or Cardiofilaria species, animal filariids that can be sympatric with B. malayi. P32-labeled clone pBm 15 correctly identified mosquitoes infected even with 1 infective larva of B. malayi. This specific DNA probe should be an invaluable tool to monitor control programs of Brugian filariasis.     

Filarial infections of Mastomys natalensis and their relevance for experimental chemotherapy

Experimental filarial infections of Mastomys natalensis, strain GRA Giessen, with Litomosoides carinii, Dipetalonema viteae, Brugia malayi (subperiodic), and Brugia pahangi were compared. Mean prepatent periods of 52, 57, 107, and 73 days p.i. were observed after subcutaneous inoculation of 40, 50, 85, and 70 infective larvae of L. carinii, D. viteae, B. malayi, and B. pahangi, respectively, in the neck region. All of the L. Carinii, D. viteae, and B. pahangi infected Mastomys showed a regularly detectable microfilaraemia. In B. malayi infections 95.5% of the animals developed parasitaemias, when the larvae had been inoculated in the neck region, whereas after groin infections only in 66.7% of the animals became patient. For both Brugia species, infections in the groin resulted in considerably lower microfilarial levels. Maximum microfilariae densities could be detected at day 120 (L. carinii) and at day 1980 (D. viteae) p.i. In the case of Brugia neck infections, the microfilarial levels increased usually until the end of the observation period, 300-350 days p.i. Worm recovery rates were 63% (L. carinii), 20.6% (D. viteae), 21.1% (B. malayi), and 31.4% (B. pahangi) of the inoculated larvae. When third stage larvae of Brugia species were inoculated in the neck region, adults of B. malayi and B. pahangi were isolated predominantly from the heart of lungs (84.4 and 78.5%, respectively). Only 12.3% of B. pahangi parasites were found in the testes; 3.4% and 18.1% were localized in the lymphatics. After inoculation of infective larvae in the groin more worms could be recovered in the testes and lymphatics, i.e. 23.4% and 14.9% (B. malayi) or 19.1% and 45.2% (B.pahangi), respectively. The results are discussed under the aspect of chemotherapeutic investigations for the evaluation of microfilaricidal, macrofilaricidal or chemoprophylactic compounds. It is concluded, that Mastomys natalensis, an animal with a broad spectrum of susceptibility for filarial infections, can be used as an alternative experimental model system, similar to that of the jird.     

Infections of Brugia pahangi in conventional and nude (athymic) mice

AKR, BALB/c and CBA/Ca and T.O. mice were completely resistant to infection with third stage infective larvae of Brugia pahangi. Third, fourth and fifth stage worms transplanted from the peritoneal cavity of jirds into the peritoneal cavity of mice continued to develop. BALB/c mice were the most susceptible of the strains tested and adult worms were obtained after each type of transplanted infection. Congenitally athymic nude mice were much less resistant to transplanted worms and infective larvae developed to full maturity in most of them. Ten of 14 athymic mice infected by the intraperitoneal (ip) inoculation of infective larvae had microfilariae in their blood or peritoneal cavities. At autopsy a percentage recovery of adult worms of 0-38% (mean 11.1%) was obtained. Microfilariae were only found in the blood of 2 of 6 athymic mice infected by subcutaneous (sc) infection and at autopsy 0-19.1% (mean 6.1%) recoveries were obtained. The thymic littermates of the nudes were more resistant than those most of the other strains used.